Symphotime

A sedimentation assay was performed to determine the polymerization state of septins by diluting septin complexes into a low salt buffer (50 mM KCl, 50 mM Tris, pH 8.0, and 1 mM DTT) for 2 h. Next, samples were centrifuged for 20 min at 22°C under 100,000 RCF (Optima Ultracentrifuge; Beckman Coulter). Supernatant was removed, and pellets were resuspended in the same volume. Samples were then analyzed by SDS-PAGE.
The FCS autocorrelation curve of fluorescent septin complexes in high salt buffer (300 mM KCl, 50 mM Tris, pH 8.0, and 1 mM DTT) was generated using commercial PicoQuant hardware and software on a Nikon A1 LSM, using a Plan Apo IR 60× WI 1.27NA objective. Identical laser intensity was used when comparing complexes containing Cdc11–SNAP–Atto488 and the mutant Cdc11-α6–SNAP–Atto488. Fluctuations in fluorescence intensity were monitored for 20 s for each experiment. The autocorrelation function was obtained with after pulsing suppression by means of fluorescence lifetime correlation spectroscopy with a pulsed 485-nM laser (40 mHz) in SymPhoTime (PicoQuant).

Confocal fluorescence imaging, FLCS, PCH and FLIM-FRET experiments were performed with a Microtime200 scanning confocal time-resolved microscope system (Picoquant GmbH) (Supplementary Figure S1); A 465 nm picosecond pulsed laser was used to excite the GFP tag and Alexa488 labeled antibodies. Cy3 was excited by a 532 nm laser for in vitro PCH calibration. The excitation beam was delivered to the sample stage through an apochromatic water immersion objective (60×, N.A. = 1.2) and the fluorescence was collected by the same objective, after which the emission was separated by a dual band dichroic (z467/638rpc for blue laser, 49004 DCXR for green laser, Chroma). A 50 μm pinhole was employed to block the off-focus photons and the final signal was additionally filtered by a band-pass filter (520 ± 20 nm for green emission, 610 ± 30 nm for red emission, Chroma) before reaching the single photon avalanche photodiode detectors (SPAD) (SPCM-AQR, PerkinElmer Inc.). Fluorescence information was recorded using the TCSPC (time-correlated single photon counting) module in the time-tagged time-resolved (TTTR) mode (TimeHarp200). Raw fluorescence images and autocorrelation data were first analyzed and then exported with the SymPhoTime software package (PicoQuant GmbH) for further processing. For detailed mathematical fitting and analysis procedure, please refer to the Supplementary Methods.

To allow visualization of the FG-Nup49 droplets, a small amount (500 nM) of fluorescently labeled FG-Nup49 was premixed with the unlabeled protein before flowing into the device. Depending on the fluorophore present on the cargo, we used FG-Nup49 labeled with either Alexa Fluor 594 or Alexa Fluor 488. For all experiments that included both cargo and NTRs, they were preincubated together for 30 min to allow formation of the import complex.
Facilitated transport experiments (Figs. 2, 4, and S3, A and B) were performed at room temperature on a custom-built epifluorescence microscope equipped with a 10× air objective (NA 0.4) and scientific complementary metal oxide semiconductor cameras (sCMOS ORCA, Hamamatsu) that allowed imaging a large field of view covering of the whole microfluidic chip to monitor the cargo accumulation over time. For experiments investigating the passive exclusion properties of the droplets (Figs. 3 and S3 C), a custom-built confocal microscope was used to obtain better sectioning. The confocal microscope is equipped with a 60× water objective (NA 1.2) and PicoQuant hybrid detectors. The acquisition software employed was custom-written in LabVIEW (National Instruments) for the epifluorescence microscope and SymPhoTime (PicoQuant) for the confocal microscope.

FCS measurements were performed on the confocal microscope detailed in the previous section. Fluorescence was detected through a 525/50 single bandpass filter (Semrock, Rochester, NY) on a single photon avalanche photodiode (Micro Photon Devices, Bolzano, Italy). Single photon events were recorded by a HydraHarp 400 time-correlated single photon counting module (PicoQuant, Berlin, Germany). Measurements were controlled with the softwares LAS AF (Leica Microsystems) and SymphoTime (PicoQuant). Acquisition time for one FCS measurement was 30 s. Time-resolved raw data were exported, and autocorrelated data were generated with the software F2COR (34 (link)). Autocorrelated data were then imported in the software QuickFit 3.0 (DKFZ, Heidelberg, Germany) and fitted in batch with an anomalous three-dimensional diffusion model.